Hidden in the fog: morphological and molecular characterisation of Derogenes varicus sensu stricto (Trematoda, Derogenidae) from Sweden and Norway, and redescription of two poorly known Derogenes species

Derogenes varicus (Müller, 1784) is widely reported as a trematode with exceptionally low host specificity and a wide, bipolar distribution. However, several recent studies have suggested that D. varicus represents a species complex and based on molecular evidence, four genetic lineages (labeled as “DV1–4”) have been designated within the D. varicus species complex. This possibility requires improved (ideally molecular) characterisation of specimens from the type-host (Salmo salar) and type-locality (off Denmark). During examination of trematode parasites of fish from Scandinavian and Arctic waters (Sweden and Norway), we found specimens of D. varicus in the stomach of Merlangius merlangus off the coast of Sweden, and in Gadus morhua off the coast of Sweden and Norway; we compared them to D. varicus from the type-host, the Atlantic salmon Salmo salar from Norway, to verify their conspecificity. Newly generated sequences (28S rDNA, ITS2 and cox1) of Scandinavian and Arctic specimens consistent with D. varicus all formed a single clade, DV1. 28S sequences of D. varicus from S. salar from Norway, i.e., close to the Danish type locality, clustered within the DV1 clade along with sequences of D. varicus from various hosts including Limanda limanda, G. morhua and Myoxocephalus scorpius from the White Sea and the Barents Sea (Russia), without any host-related structuring. We thus consider that the lineage DV1 represents D. varicus sensu stricto. Additionally, specimens from M. merlangus had a similar morphology and anatomy to those of D. varicus from L. limanda, G. morhua and M. scorpius from T. Odhner’s collection, supporting the presence of a single species in the DV1 lineage designated herein as D. varicus sensu stricto. We redescribe D. varicus sensu stricto, add new morphological characters and provide morphometric data. We infer that D. varicus types DV2–4 all relate to separate species. We also revise type-specimens of Derogenes minor Looss, 1901 from the A. Looss collection in the Swedish Museum of Natural History and provide redescriptions of it and of the type-species of the genus, Derogenes ruber Lühe, 1900. In light of their morphological distinctiveness relative to D. varicus sensu stricto, we reinstate D. parvus Szidat, 1950 and D. fuhrmanni Mola, 1912.

Several authors have questioned the reported broad distribution and the lack of host specificity of D. varicus and have suggested that D. varicus comprises more than one species [20,34].As a result, Køie [34] proposed to designate the northeast Atlantic specimens "D.varicus sensu lato" or "Derogenes sp.".That D. varicus is a species complex was confirmed recently by Krupenko et al. [35] who, based on molecular evidence, suggested the presence of four genetic lineages (designated as "DV1-4") within the species.The vertebrate hosts of the adult stages were as follows: Derogenes cf.varicus DV1 from the White Sea, Barents Sea (Russia) and North Sea (Sweden and Norway), a euryxenous species, occurring in the Atlantic wolffish Anarhichas lupus, the Pacific herring Clupea pallasii, Navaga Eleginus nawaga, the cod Gadus morhua, the common dab Limanda limanda, the shorthorn sculpin Myoxocephalus scorpius and the Moustache sculpin Triglops murrayii; Derogenes cf.varicus DV2, known only from the American plaice Hippoglossoides platessoides from the North Sea and from gastropod hosts Amauropsis islandica, Euspira pallida and Buccinum scalariforme from the White Sea and Barents Sea (Russia); Derogenes cf.varicus DV3 known only from the Sea of Okhotsk, Pacific, from Fedorov's lumpsucker Eumicrotremus fedorovi; and Derogenes cf.varicus DV4 known only from Hippoglossoides platessoides from the North Sea [35,63].
In the course of parasitological examination of trematodes of marine fish from Scandinavian waters (Sweden and Norway) and the Arctic Ocean (Norway), we found specimens resembling D. varicus in the stomach of Merlangius merlangus off the coast of Sweden, and in Gadus morhua from Sweden and Norway, hosts from which D. varicus was previously recorded in the North Sea.We compared these with D. varicus from the Atlantic salmon Salmo salar, to confirm their conspecificity.
Here, we provide a morphological redescription of D. varicus sensu stricto and confirm its lack of host specificity using molecular sequences (28S ribosomal RNA, Internal transcribed spacer ITS2, and Cytochrome c oxidase I cox1 sequences).Additionally, we studied the type-material of D. minor Looss, 1901 from the brown wrasse, Labrus merula.We designate a lectotype and paralectotypes and provide a redescription of this species.We also found in A. Looss's collections specimens of the type-species of the genus, D. ruber Lühe, 1900 for which we provide an illustrated redescription.The validity of species previously synonymised with D. varicus is considered.

Host and parasite collection
Fish were collected from Sweden, Northeast Atlantic (Skagerrak, Kattegat and Gullmarsfjorden) and from western and Arctic Norway (Table 1).
Specimens from Skagerrak and Kattegat were collected during the biannual International Bottom Trawl Survey by the SLU Aqua team as part of the International Bottom Trawl Survey along the Swedish coast within the scope of their research projects and permits.Specimens were euthanized and made available for examination.Specimens from Gullmarsfjorden were collected in the vicinity of Kristineberg Center for Marine Research and Innovation, outside of the borders of the Gullmarns nature reserve, and within the scope of the permit for animal research from the Swedish Board of Agriculture (Enheten för försöksdjur och sällskapsdjur, Jordbruksverket, Dnr.5.2.18-5483/18) and ethical approval for animal research from the Uppsala animal ethics committee (Uppsala djurförsöksetiska nämnd, Jordbruksverket, Dnr.5.8.18-17209/2021) issued to the Swedish Museum of Natural History.Specimens from the Arctic Ocean were collected during the HHUMTL22 cruise by the Arctic University Museum of Norway, within the scope of the fieldwork sampling permit issued by the governor of Svalbard (RiS-1D12021Al) and the permission to trawl from the Norwegian Directorate of Fisheries (21/16250).Digeneans were collected from freshly killed fish.The gastrointestinal tract was removed and examined for trematodes using the gutwash method [12,28].Trematodes were heatkilled, fixed without pressure in near-boiling saline, and preserved immediately in 80% ethanol for parallel morphological and molecular characterization.Ten specimens were processed as hologenophores (sensu [66]).

Morphological methods
Whole-mounts for morphological analysis were stained with acetocarmine or paracarmine, dehydrated in a graded ethanol series, cleared in clove oil, and mounted in Canada balsam.The hologenophores were processed according to the same methods.Drawings were made through a Nikon Eclipse i80  (Müller, 1784) from fish around the world.References for the records are given between parentheses.Note that this is not an exhaustive list of all reported hosts, excluding those for which evidence is partly lacking and illustrates mainly the numerous reports and the taxonomic range of hosts.Measurements of whole-mounts and of hologenophores are in micrometres and indicated as the range followed by the number of measurements in parentheses.The following abbreviation is used: SMNH, Swedish Museum of Natural History, Stockholm, Sweden.

Trees and distances
Phylogenetic analyses were performed using the newly generated sequences of D. varicus and those of closely related species available in GenBank (Table 2), mostly D. varicus complex and Progonus muelleri (Levinsen, 1881) complex provided by Krupenko et al. [35].Alignments for each gene region were constructed separately in AliView [37].The alignment was manually refined and trimmed to the shortest sequence.Nucleotide substitution models for phylogenetic analyses using the Maximum Likelihood (ML) method were selected using MEGA11 [80].The Kimura 2-parameter model with Gamma Distributed (K2+G) model was selected for the 28S, Kimura 2-parameter (K2) model for ITS2, and Tamura-Nei model (TN93) with Gamma Distributed with Invariant sites (HKY+ G+I) for cox1.All trees were constructed in MEGA11, with 500 replications.The Neighbor-Joining (NJ) method [72] was also used for comparison in MEGA11, with 2,000 bootstraps computed for cox1, ITS2 and 28S from the same datasets.P distances [31] were computed from the same datasets with MEGA11.

Molecular characterisation
The NJ and ML methods led to similar tree topologies and thus only the ML trees are shown (Figs.2-4).
A total of 800-857 bp of 28S rDNA were successfully sequenced for 11 individuals of D. varicus.The 28S dataset included 48 nucleotide sequences of derogenids.The trimmed matrix included 748 positions.The genetic divergence among the newly generated sequences was 0%.The newly generated sequences of D. varicus from S. salar from Norway and from M. merlangus from Sweden and those from G. morhua from Sweden and Norway were identical to sequences of D. varicus DV1 from G. morhua, M. scorpius, L. limanda, A. lupus, E. nawaga and C. pallasii from the White and Barents seas [35].
All newly generated sequences differed from those of D. cf.varicus DV2 from H. platessoides from the North Sea, Amauropsis islandica and Buccinum scalariforme off the coast of Russia [35] and from D. varicus from Eumicrotremus fedorovi from the North Pacific [22] by 2% (16 substitutions).The highest interspecific variation was between D. varicus DV1 and D. lacustris from Galaxias maculatus off the coast of Argentina [82], reaching 9% (68 substitutions).The divergence between D. varicus DV1 and P. muelleri 1 from M. scorpius, from L. limanda and from Triglops murrayi, and P. muelleri 2 from M. scorpius and from E. fedorovi collected off the coast of Russia [35] was 7%.
The newly generated 28S sequences of D. varicus from G. morhua, M. merlangus and S. salar collected off the coast of Sweden and Norway clustered as a well-supported clade (Fig. 2), with sequences designated as D. varicus DV1 (see [35]).This clade was well separated from the D. cf.varicus DV2 clade (from H. platessoides, Amauropsis islandica and B. scalariforme), the D. cf.varicus DV3 clade (from E. fedorovi) and the D. lacustris clade (from G. maculatus).Sequences of D. varicus sensu stricto from the type-host S. salar from Norway clustered within the D. varicus DV1 clade, without any host-related structuring, and we refer to the DV1 clade of Krupenko et al. [35] as "D.varicus sensu stricto".Another clade with high support included P. muelleri 1 (from M. scorpius, from L. limanda and from T. murrayi) and P. muelleri 2 (from M. scorpius and from E. fedorovi).The sub-clades relating to P. muelleri 1 and P. muelleri 2 were not well supported.a Two sequences are wrongly annotated on GenBank: OM761977.1 and OM762017.1,these two Derogenes varicus complex sp.DV1 isolates are in fact DV2.A total of 434-562 bp of ITS2 were successfully sequenced for the same 10 individuals of D. varicus as for 28S.The ITS2 tree was constructed using 35 sequences of derogenids (Fig. 3).The trimmed matrix included 429 positions.The newly generated ITS2 sequences of D. varicus from M. merlangus and from G. morhua from Scandinavian waters and those of D. varicus DV1 from the previously mentioned hosts were identical.Sequences of D. varicus DV1 differed from D. cf.varicus DV2 from the gastropods Amauropsis islandica, B. scalariforme and Euspira pallida off the coast of Russia [35] by 4%.
The newly generated ITS2 sequences of D. varicus from M. merlangus and from G. morhua from Scandinavian waters and D. varicus DV1 from the previously mentioned hosts clustered within a well-supported clade, separated from the D. cf.varicus DV2 clade.Within the P. muelleri clade collected off the coast of Russia [35], P. muelleri 1 subclade (from M. scorpius, from L. limanda and from T. murrayi) was well supported relative to the P. muelleri 2 subclade (from M. scorpius).
All newly generated cox1 sequences of D. varicus clustered with those of D. varicus DV1 within a well-supported clade (Fig. 4), well separated from that of D. cf.varicus DV2 and from the D. lacustris clade.Another clade with strong support included two sequences of P. muelleri from M. scorpius which nested as sister clade to the Derogenes clade.Type-host: Salmo salar (Salmoniformes: Salmonidae), the Atlantic salmon [58].

Morphology
Type-locality: off the coast of Denmark, Northeast Atlantic [58].
Specimens deposited: Specimens with molecular information: anterior parts of specimens mounted on slide, posterior part used for molecular analysis: specimens from Merlangius merlangus off the coast of Sweden Pre-oral lobe short, surmounting oral sucker.Oral sucker rounded.Prepharynx absent.Pharynx small, muscular, subglobular to stocky.Oesophagus short.Intestinal bifurcation in anterior third of forebody.Intestinal caeca terminating blindly posteriorly to vitelline masses.Ventral sucker round, voluminous, generally as long as wide, located at level of mid-body.
Testes globular, symmetrical to slightly oblique, immediately posterior to ventral sucker.Seminal vesicle oval, muscular and thin-walled, located usually anterior to ventral sucker with position affected by state of contraction of specimens.Pars prostatica relatively long, difficult to trace in unflattened specimens, lined by numerous gland cells, leading to seminal vesicle.Sinus-sac small, oval.Ejaculatory duct and metraterm visible passing into sinus-sac then into sinus-organ forming hermaphroditic duct.Hermaphroditic duct tubular thin-walled.Sinus-sac present.Cone-shaped permanent muscular sinusorgan projecting into genital atrium.Genital pore transversely oval, ventral, posterior to pharynx.
Ovary oval, posterior to ventral sucker, generally overlapping right testis or/and right vitelline mass.Laurer's canal not observed.Vitelline masses oval, paired, posterior to ventral  sucker, situated on each side of body, symmetrical to oblique.Vitelline ducts joined medially posteriorly to ovary, forming thick common duct.Seminal receptacle oval, small.Uterus convoluted, with coils extending posteriorly between vitelline masses, passing dorsally to ventral sucker, barely visible as straight thin-walled tube, entering sinus-sac forming metraterm.Eggs elongate, oval.Excretory vesicle Y-shaped, bifurcating just behind vitelline masses; branches reuniting dorsally to pharynx.
Remarks: We found specimens in T. Odhner's collections from G. morhua, L. limanda and M. scorpius that we reexamined.We attempted to detect any host-induced variations.Morphometric data are presented in Table 3. T. Odhner's specimens from these three hosts share with our specimens of D. varicus the sausage-shaped body appearance and the organisation of the terminal genitalia.They differed by body length, forebody and hindbody length, ventral sucker size and size of the seminal vesicle.However, the number of measured specimens is low (one from L. limanda, 2 from M. scorpius, and 2 from G. morhua).In addition, from our examination, it is clear that T. Odhner flattened his specimens, which impairs morphometric comparison [12,25].However, we think it is likely that specimens collected by T. Odhner from L. limanda, M. scorpius, and G. morhua from Kristineberg are conspecific with our newly collected material from the Swedish coast of Skagerrak and off the coast of Norway, given the general morphological similarities and collection localities.Type-host: Chelidonichthys lastoviza (Bonnaterre) (Perciformes: Triglidae) (as Trigla lineata Gmelin), the Streaked gurnard [47].
Site in host: Intestine (present paper).Material examined: one slide (SMNH-138743) containing two adult specimens (originally numbered by A. Looss as 1818) of which one matches an unpublished line drawing of D. ruber (Fig. 9) found in A. Looss's archive, collected from C. lastoviza on 12. 1900 in Trieste, Italy, Western Mediterranean (corresponding measurements of this specimen presented in Table 4) and four unpublished line drawings of D. ruber from Scorpaena scrofa Linnaeus (Fig. 11).
Ovary post-testicular, in anterior half of hindbody, situated at considerable distance behind ventral sucker, obscured by eggs.Uterine coils tightly packed with eggs, reaching posterior body end, distributed along entire hindbody; in forebody, uterine coils starting from anterior level of ventral sucker to short distance behind terminal genitalia.Metraterm muscular, joining male ejaculatory duct.Vitelline masses postovarian, paired, lobed.Eggs numerous, mostly collapsed in hindbody.Excretory vesicle not observed due to coiling of body posteriorly.Excretory arms visible in forebody; position of bifurcation of excretory arms not observed.
Remark: Derogenes ruber Lühe, 1900, the type-species of the genus, was first described from the tub gurnard Ch. lastoviza off the coast of Split, Croatia [47].Being known only by the original description that lacked illustrations, we here provide a figure and additional information especially regarding the organisation of the terminal genitalia, based on two specimens found in Looss's collection from the same host from off Trieste, Italy.Looss's archive also included four drawings of D. ruber    from Scorpaena scrofa Linnaeus (Fig. 11) that we present in Figure 12.The corresponding slides could not be found in Looss's collection.
Archival documents: The archives include the original line drawing published as Figure 5 by Looss (1901), and an unpublished ink drawing of the eggs and of the terminal genitalia (see Fig. 13 here).
Testes oval, near left and right margins of body posterior to ventral sucker.Seminal vesicle tubular, placed at short distance anteriorly to ventral sucker.Seminal vesicle followed by long pars prostatica, lined by small unicellular prostatic glands.Ejaculatory duct and metraterm visible passing into sinus-sac and forming hermaphroditic duct.Hermaphroditic duct thin, enclosed in sinus-organ, projecting into genital atrium (Figs.14B and  14C).Genital pore opening medially, posteriorly to pharynx.
Ovary in hindbody, sinistral, close to body margin, slightly larger than testes.Seminal receptacle long, tubular, thin-walled, immediately anterior to vitelline masses.Laurer's canal not observed.Vitelline masses postovarian, paired, compact, separated from one another.Excretory bladder Y-shaped: arms of excretory bladder reaching midlevel of pharynx.Eggs oval, thin shelled.
Remark: Derogenes minor is currently considered a valid species (WoRMS, 2022).The redescription here was based upon one slide (SMNH-138741) containing two adult specimens of which one matches the original description and illustration given by Looss (1901) (see his Fig. 5).Additionally, the sketching of D. minor found in A. Looss's archive (see Fig. 13 here) matches the figure given in the original description as collected on 27.09.1900 off the coast of Trieste, Italy.This slide is thus designated as the Lectotype.Additional material, specimens from Labrus merula preserved in ethanol (SMNH 892) currently in the wet collections of the SMNH, are designated as Paralectotypes.Additional specimens include one specimen labelled as D. minor by A. Looss, from the Intestine of Lophius piscatorius Linnaeus, collected on 12.1900 off Trieste, Italy (SMNH-138742); one specimen labelled as Derogenes sp. by A. Looss, from the Intestine of Lophius piscatorius Linnaeus, collected on 10.1904 off Trieste, Italy (SMNH-208362) is designated here as D. cf.minor (Fig. 14D).

Delimitation of D. varicus sensu stricto
Derogenes varicus has been considered the most common digenean species in fish [32].It is widely reported in most of the oceans of the world as well as in freshwater systems.In the Northeast Atlantic, it has been recorded from more than 40 species of marine fish [49].There are also some freshwater records, mainly in migratory diadromous fishes, such as salmonids [20].The life cycle was described in detail by Køie [32]; naticid snails are the first intermediate hosts, releasing cystophorous cercariae that can infect calanoid copepod, among which only Calanus spp.may act as second intermediate hosts.
In definitive hosts, the adults are usually found in the oesophagus or stomach.Several authors have suggested that D. varicus comprises two or more species and Køie [34] suggested that D. varicus from the northeast Atlantic should be referred to as D. varicus sensu lato or as Derogenes sp.A recent comprehensive study by Krupenko et al. [35] provided evidence that D. varicus can be genetically split.Four genetic groups were recognised, independent of locality or host to a certain extent: D. varicus DV1 occurring in G. morhua, M. scorpius, L. limanda, A. lupus, E. nawaga and the Pacific herring C. pallasii, D. cf.varicus DV2 from H. platessoides, D. cf.varicus DV3 from E. fedorovi Mandrytsa, 1991, and D. cf.varicus DV4 from H. platessoides (based on 18S sequences).Herein, all the newly generated 28S sequences of D. varicus from fishes collected off the coast of Sweden and Norway, including that from a specimen from the type-host S. salar were identical, and clustered within the D. varicus DV1 clade.Although the typelocality of D. varicus is Danish marine waters, our collections off the coast of Sweden essentially encompass this area.Additionally, Müller [58] did not specify a locality from which he collected the salmon infected by "Fasciola varica".He mostly worked in Øresund in Denmark and in Drøbak in the Oslofjord, Norway.It seems most likely that the studied salmon originated from the Øresund salmon fishery.Thus, we consider the clade identified as D. varicus DV1 by Krupenko et al. [35] to represent the true D. varicus, herein designating it as "D.varicus sensu stricto".
Our D. varicus sensu stricto specimens from M. merlangus were morphologically similar to that of D. varicus sensu lato from L. limanda, G. morhua and M. scorpius from T. Odhner's collection, which supports the presence of a single species in the D. varicus DV1 lineage.More importantly, the intraspecific genetic variations between members of the D. varicus DV1 (i.e. from different host species) were of an order of magnitude lower than the interspecific divergence between members of D. varicus DV1 and the well established species D. lacustris: 0% vs. 8-9% (68 substitutions) in 28S, 0-1% vs. 19% in cox1.The divergence of cox1 reported herein for D. varicus sensu stricto agrees with intraspecific variations, which are typically below 6.0% in Digenea [82] and the genotypes were independent of host species or localities.Among Derogenidae, previously reported interspecific divergences in cox1 ranged between 10.5-15.1% [83] and 16.9-20.4%[83], while in other lineages divergence reached 8.9-26.5% [6]; 9.6-12.8%[4]; 9.9-15.1 [44]; and 20% [26].Hence, divergences reported within the DV1 clade appear consistent with intraspecific divergences.Consequently, we consider the lineages DV2, DV3 and DV4 to represent different species.In light of the available data, D. varicus sensu stricto (DV1) is euryxenous.Hence, this work is a step towards untangling this species complex and delimiting the cryptic complex hidden under the single name "D. varicus".
We compared our D. varicus sensu stricto from S. salar, M. merlangus and from G. morhua from Sweden and Norway, with specimens from T. Odhner's collection (Table 3).However, T. Odhner's specimens were excessively flattened and thus to consider any differences as intraspecific variations is unreliable.Additionally, they had similar morphology and anatomy (Figs. 7 and 8).Derogenes varicus from M. merlangus and from G. morhua from Scandinavian waters are genetically identical to D. varicus sensu stricto from various hosts (see [35]).They were also morphologically similar as measurements overlapped (Table 3).Additionally, they showed no divergence in 28S or ITS2 sequences and differed only by 0-1% in the cox1 gene region.
Problems arise with species identification within the D. varicus complex because several species of Derogenes, including the type-species, were described only superficially [47], and data on taxonomically important characters of the "true" D. varicus were incomplete.The original description of D. varicus does not allow consideration of several important morphological characteristics such as the extent of the intestinal caeca, position of the genital pore, relative position of the ovary and testes, and several other features, some of which are now used to distinguish Derogenes species.Consequently, the redescriptions and delimitation of D. varicus sensu stricto given herein are crucial for untangling the species complex in the future to describe and discriminate the cryptic species detected in D. varicus sensu lato.

Other species within Derogenes
The type-species of the genus, D. ruber Lühe, 1900, was first described from the gall bladder of the streaked gurnard Ch. lastoviza (as the syn.Trigla lineata) from off the coast of Roving, Croatia, Adriatic Sea [47].It was redescribed from the piper gurnard Trigla lyra from off the coast of Split, also Croatia [74].We noted some minor morphometrical differences between the two Adriatic records: Derogenes ruber from T. lyra differed from D. ruber from the type-host Ch. lastoviza by having a smaller body (4200-4500 Â 1300-1800 vs. 5000-6000), smaller ventral sucker (950-1290 vs. 750), smaller pharynx (168 vs. 200) and ovary (252 vs. 450).However, the number of measured specimens is low (2 specimen were measured in both studies) [47,74].Similarly, specimens of D. ruber from Ch. Lastoviza from Italy examined herein differed slightly from those from the same host, from the type locality Croatia, by having a larger body (7869 Â 1847 vs. 5000-6000 Â 2000), larger oral sucker (655 Â 708 vs. 505) and larger pharynx (200 vs. 168).We also note that despite sharing the same host, Ch. lastoviza, D. ruber of Lühe [47] were collected from the gall-bladder while the two specimens examined here were collected from the intestine.We do not know if this is a postmortem migration of D. ruber as the gall-bladder and the intestine (and also pyloric caeca) were regarded as unusual sites for derogenids, which are normally stomach parasites [2].Additionally, D. ruber from T. lyra reported by Sey [74] and from the present specimens by having strikingly smaller eggs (23 Â 23).However, Bartoli and Gibson [2] pointed that this egg-size value is presumably an error.
A species very similar to D. ruber is D. latus Janiszewska, 1953, first described based on a single specimen from the intestine of the red mullet Mullus barbatus from the same Adriatic locality as that of D. ruber, off Split, Croatia [27].
Derogenes latus was redescribed from the intestine of M. barbatus and Trisopterus capelanus (Lacépède, 1800) in the North Adriatic Sea [64] and from the gall-bladder of M. surmuletus off Corsica (France), Western Mediterranean [2].The redescription provided by Bartoli and Gibson [2] based on accessible voucher material and serial sections provided  several morphological and anatomical details along with morphometrical data.Derogenes latus was frequently reported from its type host from the Western Mediterranean, off Spain [16] and off France [38], and from a closely related host, M. surmuletus from the Western Mediterranean (off France and Algeria [7,23,38,81]).
Curiously, we found in Looss's archive unpublished lined drawings of derogenids identified as D. ruber, from S. scrofa, mentioned also as a host for D. latus [46] and off France [81].We could not find the slides used to make these illustrations.Despite having different hosts, C. lastoviza for D. ruber [47] and M. barbatus for D. latus [27], the distinction between D. ruber and D. latus has been questioned [2].Derogenes ruber and D. latus share a stout body, post-testicular vitellarium composed of two multilobed masses and a uterus occupying almost the entire body [2,64,68].We have confirmed these features in specimens from A. Looss's collections, and illustrations of Derogenes ruber and D. latus clearly share the previously mentioned anatomical features.
Although recent studies have used molecular sequence data to test the putative broad host specificity of some species of Derogenes and both D. lacustris [82] and D. varicus sensu stricto have been shown to occur across a wide variety of hosts (see Figs. 2-4), as mentioned above, even if D. ruber is known only from its type host, a Triglidae (Perciformes), the closely related species D. latus was reported from 6 species, belonging to 5 families (Mullidae, Scorpaenidae, Sparidae, Dorosomatidae, and Phycidae) across 5 orders (Mulliformes, Scorpaeniformes, Spariformes, Clupeiformes, and Gadiformes) and consequently, synonymising the two species without supporting molecular data will make D. ruber a euryxenous species occurring in hosts across 7 families and across 6 orders.Such a nomenclature act without supporting molecular data might lead to making D. latus the Mediterranean version of "D.varicus".Although Bartoli and Gibson [2] highlighted marked morphological similarity between D. latus and D. ruber and convincedly argued about possible synonymy between the two species, they refrained from synonymising the two species formally until further studies of material from the type-hosts and localities are available.Molecular data of D. ruber have recently been made available [18], but we refrain from synonymising the two species until molecular data are available for D. latus.
Hence, in light of available data, we can only affirm that D. ruber and D. latus Janiszewska, 1953 are very similar, apparently differing only in the degree of lobation in the vitelline masses.They do differ in principal host and site and taking into consideration the potential synonymy between the two species suggested (Bartoli and Gibson [2]), molecular studies on new material are needed to clarify this issue.Additionally, we could unfortunately not examine any D. latus and thus the validity of this species cannot presently be further considered.
Derogenes minor, currently a valid species [21], was first described from the brown wrasse Labrus merula (Labridae) from off the coast of Trieste, Italy [45].Although some ambiguity surrounded the collections of A. Looss after leaving Egypt at the beginning of World War I, part of his collections was sold to Dr. Theodor Odhner, a former Professor at the Department of Invertebrate Zoology at the Museum of Natural History (Naturhistoriska riksmuseet) in Stockholm by his widow [36,62].The archive of the SMNH includes a list of material of A. Looss, including hosts, locality, date, publication-ready ink drawings and unpublished line drawings.The specimens of D. minor we found in A. Looss's collection agree with the original description and one of the two specimens examined matches the line drawing found in the archive of A. Looss and the drawing published as Figure 5 by Looss (1901).Hence, this is clearly the type-material of D. minor.We here supplement the original description [45] by providing additional morphometrical data and with a description of the organisation of the terminal genitalia based on re-examination of type specimen.
In A. Looss's collection, we also found two putative specimens of D. minor from the intestine of the angler, Lophius piscatorius, caught off the coast of Trieste, Italy.One of them lacking the posterior part is labeled as D. minor and the second one is entire and labelled by A. Looss as Derogenes sp. (Fig. 14D).Measurements of D. minor from L. piscatorius and those we examined from L. merula overlapped.The specimen labelled as Derogenes sp. from L. piscatorius differed slightly from D. minor from the type-host L. merula by having a larger body and larger vitelline masses.They shared, however, the lobed vitelline masses, specifically the mulberry-like shape, a characteristic of D. minor [45].This situation is puzzling as L. piscatorius was reported as a host of D. latus Janiszewska, 1953 [2,3].However, D. latus and D. minor are supposed to be distinguished partially by the shape of the vitelline masses, being mulberry-like in D. minor [45] vs. deeply lobed or lobed in D. latus [27].The vitelline masses of the specimen we examined from L. piscatorius (Fig. 14D) appear intermediate between the previously mentioned shapes; it appears mulberry-like with deep lobes but resembles D. minor in its narrow hindbody.Hence, as we examined only one specimen, on which unfortunately the terminal genitalia could not be observed, we identify the derogenids examined from L. piscatorius as D. cf.minor pending further study.
A diagram comparing four species of Derogenes (Fig. 15) suggests that the organisation and shape of vitelline masses is the most straightforward character to distinguish Derogenes species.In D. varicus sensu stricto, the vitelline masses are unlobed (Fig. 15A).In D. ruber, the vitelline masses are lobed and rosette shaped (Fig. 15B).In D. minor, the vitelline masses are mulberry-like shaped (Fig. 15C).In D. latus, the vitelline masses are deeply lobed, and rosette shaped (Fig. 15D).We also attempted to test the utility of the terminal genitalia, and in D. varicus sensu stricto, the sinus-sac is rounded (Fig. 15A) and the genital atrium is almost half the length of the sinus-sac, and the basal musculature of the sinus-sac does not reach the level of the genital atrium.In D. ruber, the sinussac is ovoid and elongated transversally (Fig. 15B), and the genital atrium is two-thirds of the length of the sinus-sac, and the musculature is weakly developed and extends only to the base of the genital atrium.In D. minor, the sinus-sac is rounded (Fig. 15C), and the genital atrium is small, only about one third the length of the sinus-sac and the musculature covers the genital atrium.However, the three species share a conical sinus-organ and most importantly, the previously mentioned differences are high, likely affected by the wall musculature contraction, and should be taken with caution when distinguishing species.Unfortunately, we could not examine specimens of D. latus to comment further on the form of the terminal genitalia.Hence, it would be valuable to investigate these features, especially the form and size of the sinus-organ, to test their utility in species delimitation.Although the striking morphological similarities between D. latus, D. ruber and D. minor suggests possible synonymy between the three [2], especially with respect to the large host spectrum for D. varicus supported here with molecular data, we refrain from making any taxonomic proposals regarding the Mediterranean specimens as molecular data are lacking and we consider them valid species pending further studies.The following species have previously been synonymised with D. varicus: Anisocoelium hippoglossi MacCallum, 1921, D. fuhrmanni Mola, 1912 and D. parvus Szidat, 1950 [85].Anisocoelium hippoglossi MacCallum, 1921, described from the Atlantic halibut Hippoglossus hippoglossus (Linnaeus, 1758) collected off the coast of Massachusetts, USA exhibits unique characters relative to D. varicus sensu stricto, with the ovary a considerable distance in front of the ventral sucker [48].If these features were correctly characterised, then A. hippoglossi MacCallum, 1921 is not a synonym of D. varicus sensu lato nor even a species of Derogenes.Since the species appears to never have been recollected, it could be that the seminal vesicle of a derogenid was taken for the ovary, or that the specimen was teratological.Derogenes fuhrmanni Mola, 1912, was first described from the European bullhead Cottus gobio Linnaeus from River Aniene, in Lazio, Italy and additional specimens brought from Rome as Mola stated "In etwa 30 Exemplaren von Cottus gobio, wovon einige aus dem Flusse Aniene stammten und andere von mir in Rom" [56].It was subsequently synonymised with D. varicus [67].are far smaller in D. parvus than those described by Looss in D. minor [79].We therefore consider D. parvus a distinct and valid species.

Figure 1 .
Figure 1.Geographical distribution of Derogenes varicus(Müller, 1784) from fish around the world.References for the records are given between parentheses.Note that this is not an exhaustive list of all reported hosts, excluding those for which evidence is partly lacking and illustrates mainly the numerous reports and the taxonomic range of hosts.

Figure 2 .
Figure 2. Tree inferred using the ML method based on the 28S rDNA sequence data; only bootstrap values higher than 70 are indicated.The newly generated sequences are indicated in red.Sequence of Derogenes varicus from the type-host is in bold.Lineages DV1, DV2, DV3 and Derogenes lacustris are in different colours.

Figure 3 .
Figure 3. Tree inferred using the ML method based on the ITS2 rDNA sequence data; only bootstrap values higher than 70 are indicated.Derogenes varicus lineages DV1, DV2 are in different colours.

Figure 4 .
Figure 4. Tree inferred using the ML method based on the cox1 gene sequences; only bootstrap values higher than 70 are indicated.The newly generated sequences are indicated in red.Lineages DV1, DV2 Derogenes lacustris Tsuchida, Flores, Viozzi, Rauque & Urabe, 2021 are in different colours.

Figure 13 .
Figure 13.Derogenes minor Looss, 1901 ex Labrus merula.Original line drawing of D. minor published as Figure 5 in Looss (1901), and an unpublished line drawing of the terminal genitalia.
In addition to the hosts being different (Cottus gobio, Cottidae for D. fuhrmanni vs. Salmo salar, Salmonidae for D. varicus) and especially the localities being widely separated (River Aniene, Italy for D. fuhrmanni vs. Denmark for D. varicus), Derogenes fuhrmanni also differs from D. varicus by having a larger body (4000 Â 1000 vs. 2276 Â 528), larger ventral sucker (600 vs. 353 Â 374), and strikingly larger eggs (72 Â 27 vs.40-53 Â 28-35).We consider thus D. fuhrmanni as a valid species.We also examined the original description of D. parvus first described from the Patagonian blennie Eleginops maclovinus (Cuvier, 1830) off the coast of Tierra del Fuego, southeastern Atlantic [79].It can be distinguished from D. varicus sensu stricto by the testes, the ovary, and the globular parts of the vitelline masses being all the same size, and by having a much larger ventral sucker (697 vs. 300 Â 299).The most distinctive feature distinguishing D. parvus and D. varicus sensu stricto is the vitelline masses being divided into numerous small lobes in D. parvus [79].The possession of lobed vitelline masses might suggest possible synonymy with D. minor or with D. latus and D. ruber.However, Szidat (1950) stated that the vitelline masses

Table 1 .
Host fishes examined from Scandinavian waters of the North Sea and the Arctic Ocean during this study.
C. Bouguerche et al.: Parasite 2023, 30, 35 microscope with DIC (differential interference contrast) and a drawing tube.Drawings were scanned and redrawn on a computer with Adobe Illustrator 2022.
DescriptionBased on 23 specimens.Measurements in